2016人源化实验动物模型国际论坛

南京大学—南京生物医药研究院

南京钟山医药科技转化研究院

江苏省实验动物协会

 

 

 

 John Bial 

President and Chief Executive Officer 

Yecuris Corporation

Presentation Title:

Humanized Animal Models: Novel Tools for Discovery Research

Abstract:

Animal models xenografted with human primary cells or genetically modified to constitutively express human proteins have been utilized in the fields of drug metabolism, toxicology, infectious disease, and gene therapy.  In the field of liver biology, primary cell xenografts have been successfully and reproducibly generating models with better than 95% hepatic replacement, yielding models that have a broad range of utility that are able to provide data that more closely resemble what we would expect to see in the clinic.

The FRG® KO mouse model has been widely utilized in these applications - in this talk, we will explore the mechanism of these models as well as their uses and limitation

 

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Neal Goodwin , Ph.D.

Vice President

Corporate Research Development

Champions Oncology, Inc.

Presentation Title:

Humanized Mouse Systems for Oncology Clinical Advancement

Abstract:

The development of IL2 receptor gamma chain null (IL-2Rγnull) mice; examples of which include NOG, NSG, and MISTRG; has led to the implementation of human immune system (HIS) and human AML leukemic stem (LS) cell humanized pharmacology systems for use in oncology therapeutic development. Advanced variants of IL-2Rγnull mice with human transgenic GMCSF and IL3 have allowed for HIS mice to be created that have activated cells of myeloid lineage such as dendritic cells and macrophages. HIS mice co-engrafted with patient derived xenografts (PDX) conceptually provide for an excellent system for elucidating efficacy and mechanism of action for immune checkpoint inhibitors and for determining immune checkpoint inhibitor synergy with standard of care agents. A drawback to HIS modeling is that the source of hematopoietic stem cells required for creating HIS mice is never a HLA match to the PDX tumor model being tested. Feasibly, only by creating autologous pluripotent hematopoietic stem cell lines that match each PDX tumor or the use of autologous hematopoietic stem cells can allow for HIS-PDX mice screens that closely resemble the natural human immune system. Approaches for the co-clinical use of HIS and LS mouse systems for co-clinical modeling and pilot data from these efforts will be presented.

 

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Gavin Thurston , Ph.D.

Vice President of Regeneron Pharmaceuticals

Presentation Title:

Genetically engineered mouse models to test human cancer immunotherapies

Abstract:

In vivo tumor models are essential to test the anti-tumor activity and side-effect profile of novel immunotherapeutics. However, antibody-based therapies often do not cross react with the corresponding murine target, making it difficult to develop relevant preclinical tumor models. We have utilized Regeneron’s capabilities in genetic engineering of mice to develop several different types of mouse tumor models with relevant immune cell targets, and have used these models for preclinical testing of both checkpoint inhibiting antibodies and T cell engaging bispecific antibodies.

 

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An Tang , Ph.D.

Project Manager, PDX platform,  NBRI

Presentation Title:

Applications of  NCG mice in Translational Cancer Research

Abstract:

Most of the oncology compounds entering clinical trials failed to demonstrate sufficient safety or efficacy. Hence, better preclinical evaluating systems which could represent the complexity of human cancer are needed. The patient-derived xenograft (PDX) models are increasingly used in translational cancer research and have become the most realistic model for preclinical studies. By using the immunodeficient NCG (NOD-Prkdc^em26Il2rg^em26Nju) mice, NBRI has established a collection of PDX models covering gastric, colorectal, pancreatic, cervical and ovarian cancer and a collection of CDX models covering scores of cancer cell lines. We are establishing an Oncology platform, together with the Phenotyping platform and Pathology platform, to facilitate translational cancer studies and provide services in oncology drug screening, biomarker development and personalized medicine strategy evaluation. 

 

免疫专题

   

 

Zhigang Tian , Ph.D.

Institute of Immunology

School of Life Sciences and Medical Center

The Key Laboratory of Innate Immunity and Chronic Disease(Chinese Academy ofSciences)

University of Science and Technology of China

Presentation Title:

Human NK Cell Development in Humanized Mice

Abstract:

Natural killer (NK) cells are important effector lymphocytes of innate immune system and distributed in various organs, such as the liver, lung, spleen and lymph nodes. They have been shown to play critical roles in defending against virus and tumors. Although NK cells are conserved in several species, many differences exist between human and mice. The study of NK cells in vivo is largely restricted to mice, the knowledge of which cannot be fully used in human NK cell biology. Humanized mice provide a powerful animal model for dynamically studying the reconstitution, development and function of human NK cells in vivo. Several humanized mice models have been constructed, but the development, maintenance and function of human NK cells are limited if compared with B- and T-lymphocytes. Because there are discrepancies between human and mice cytokines, various strategies have been used to supply different human cytokines in the humanized mice for improving human NK cells reconstitution. By these methods, the level of reconstitution, terminal differentiation and function of human NK cells are increased remarkably. A humanized mice model with fully functional NK cells will be useful for investigating the biological and pathological functions of human NK cells in vivo.

 

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Zheng Hu , Ph.D.

Associate Professor

The Academy of Translational Medicine of First Bethune Hospital,

Jilin University

Presentation Title:

Humanized mice as a novel preclinical model for translational research

Abstract:

Small animal models with functional human lymphohematopoietic systems are highly valuable for the study of human immune function and human immune relevant diseases under physiological and pathological conditions. Over the last two decades, numerous efforts have been devoted towards the establishment of such humanized mouse models. Several years ago, we firstly developed a humanized mouse model with reconstitution of almost all lineages of human immune cells, including human T cells, human B cells, human myeloid and plasmocytoid dendritic cells, through co-transplantation of human CD34+ fetal liver cells (i.v.) and human fetal thymic tissues (under renal capsule) on immunodeficient mice, which also named as BLT mouse model. Moreover, human immune system in the humanized mice are functional as both antigen specific human T cell responses and antigen specific human T cell dependent antibody production were determined in these animals after immunization, making it widely used to study issues like HIV infection, allogeneic/xenogeneic rejection, autoimmune diseases, function analysis of antibodies target human immune cells and so on. Other than human lymphoid cells, high level of human red blood cells and human platelets could also be achieved in the optimized humanized mice. Thus it may also serve as a powerful platform to evaluate novel therapeutic regimens for human hematopoietic disorders and the diseases like malaria infection. This presentation is focus on the reconstitution of human lymphohematopoietic system in these humanized mice and the potential use of these animals in translational biomedical research.

 

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Takeshi Takahashi , Ph.D.

Chief, Immunology Laboratory, Gene Manipulation Laboratory

Central Institute for Experimental Animals, Kawasaki, Japan

Presentation Title:

Generation of 2ndgeneration NOG mice and application to human immunology

Abstract:

NOD/scid/IL-2R cnullmouse (NOG), which was established in CIEA, has enabled long-term engraftment of human derived materials in mice. Transplantation of human hematopoietic stem cells (HSC) in NOG mice leads to stable reconstitution of human immune systems mainly consisted of B and T lymphocytes. CIEA has produced a series of 2ndgeneration NOG mice by introducing genetic modifications in order to improve the quasi-human immune systems. These include enhancement of multi-lineage hematopoiesis, induction of antigen specific immune responses, or recapitulation of some pathological aspects of human diseases. I would like to introduce some of the 2ndgeneration NOG mice and their immunological features. For example, expression of IL-2 or IL-15 favored the predominant differentiation of mature human NK cells. IL-3/GM-CSF or IL-6 transgenic (Tg) mice promoted the myeloid cell differentiation, which is otherwise ineffective in NOG mice. We have attempted to transplant human tumor cell lines into those humanized ‘next generation NOG mice’ to examine whether these mice become useful models for studying tumor immunology and also for developing clinical drugs. Our results demonstrated that human NK cells in IL-2 or IL-15 Tg mice could suppress the tumor growth either by graft versus leukemice (GVL) effect or by ADCC.  In addition, a significant number of immature myeloid cells, which are phenotypically similar to MDSC or TAM, could be detected in IL-3/GM-CSF or IL-6 Tg mice, We would like to present our recent results and discuss about the future direction of next generation humanized mice.

 

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Yan Li, Ph.D.

Institut Pasteur, France 

Presentation Title:

Novel human immune system (HIS) mouse models for human antibody discovery and vaccine development

Abstract:

In the past 10 years, progress in the development of ‘humanized’ mice bearing human immune systems (HIS mice) has been significant. It is now possible to generate mice with robust, stable human hematopoiesis that includes not only multiple lymphocyte subsets but also diverse myeloid cell lineages. Nevertheless, eliciting antigenRspecific adaptive immunity, especially T cellRdependent highR affinity antibody responses in current HIS mice remains challenging. 

To further optimize functionality of HIS mice, our laboratory has focused on improving three critical checkpoints required to mount efficient antibody responses. These include 1) intraRthymic and peripheral T cell selection on human HLA molecules; 2) peripheral T cell activation via human dendritic cells (DC); and 3) B cell R T cell interactions in the lymph node. The inherent limitations in previous HIS mouse models were individually corrected following specific genetic modifications to the basic Balb/c Rag2R/R γcR/R SirpRNOD (BRGS) model. Enhanced human T cell development and function, functional peripheral human DC subsets, and systemic lymph node development were achieved in a series of ‘second generation’ BRGS HIS mice that produced specific T cell response and antigen specific antibodies after immunization with model antigens. As a result, optimized BRGSRbased HIS mice offer an ideal model for human antibody discovery and vaccine development. These novel HIS mouse models can be exploited in partnership with the spinRoff company AXENIS (Paris, France) that specializes in HIS mouse technologies for the public and private sector.

 

肝脏专题

   

 

 

Hiroshi Suemizu , Ph.D.

Director, Biomedical Research Department

Central Institute for Experimental Animals

Presentation Title:

Development of advanced humanized liver model with new TK-NOG mice

Abstract:

To address interspecies difference issues, we developed animal models with reconstituted livers containing human hepatocytes (humanized-liver). This model (TK-NOG) was based on immunodeficient NOG mice that express the herpes simplex virus thymidine kinase type 1 gene (HSVtk) specifically in the liver by using an albumin gene artificial enhancer/promoter. The humanized-liver showed zonal position-specific enzyme expression and a global gene expression pattern representative of the mature human liver. However, TK-NOG mice have the following general disadvantages; 1) gender differences in transgene expression, 2) insufficient transgene expression levels at the time of weaning, and 3) male sterility. To overcome these disadvantages, we developed new TK-NOG mice that have transthyretin promoter driven HSVtk mutant clone 30 (TKmut30) transgene. We established three transgenic TK-NOG lines (NOG-TKmut30). Male NOG-TKmut30 transgenic mice in each line were fertile and reproduced normally, transmitting the transgene in expected Mendelian ratios. Male and female NOG-TKmut30 mice aged 5 weeks showed severe liver injury by ganciclovir injection. The new TK-NOG lines were also successfully engrafted with human hepatocytes as in traditional TK-NOG mice. However, it is still insufficient for complete humanized model; that is 1) affecting drug metabolizing enzyme activities by remaining mouse hepatocytes, 2) lacking inflammatory responses. In this presentation, I would like to introduce new platform of TK-NOG mice and our perspectives for immunocompromised “humanized model” especially in drug metabolism research and infectious disease research fields.

 

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Feng Li,  Ph.D.    
University of North Carolina at Chapel Hill, USA

Presentation Title:  

To improve human hepatocyte reportulation to 100% in a second generation mouse model.

Abstract: 

The repopulation of human hepatocytes is always incomplete when the balance of transplanted human hepatocytes and residual mouse hepatocyte functionally reaches in Fah Knockout mouse. To further increase the repopulation level of human hepatocytes, we produce a second generation of NRG-FAH-AFC9 mouse, which containing AFC9 transgene on top of Fah knockout. The AFC9 transgene mouse, containing a FKBP-Casp9  transgene specifically over-expressed in mouse liver,  can functionally activate the apoptosis pathway in the presence of a chemical inducer, which facilitates the complete removal of mouse cells hepatocytes. The repopulation level of human hepatocytes can reach almost 100% in liver, efficiently supporting HBV, HCV infection. The high level of human hepatocytes in mice can be potentially used for drug development.   

 

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Ludi Zhang , Ph.D.

Institute of Biochemistry and Cell Biology (IBCB),

Shanghai Institutes for Biological Sciences (SIBS)

Presentation Title:

Generation of liver-humanized animals with induced human hepatocytes

Abstract:

Humanized animals with livers reconstituted of human hepatocytes are the ideal models to study human liver disease and drug development in vivo. However, the procurement of human hepatocytes is a major problem in producing liver-humanized animals because of the shortage of donor organs. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs and display functions characteristic of mature hepatocytes. More importantly, upon transplantation into mice with fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHep cells repopulated 4.2% of the liver parenchyma and recused mice, suggesting it can be applied as an alternative cell source for generating liver-humanized animals. Notably, it has been reported that hepatocyte proliferated more efficiently in rat livers than in mouse livers and extensively liver-humanized rats have not been reported yet. Therefore, we aimed to generate a novel rat model to improve the in vivo repopulation of hiHep cells. Using lately developed CRISPR/Cas9 technique, we generated Fah^-/-Rag2^-/-IL2rg^-/- (FRG) rats. Transplantation of wild-type rat hepatocytes rescued FRG rats from the death via efficient liver repopulation. Furthermore, xenograft of human liver cancer cells and induced pluripotent stem cells in FRG rats demonstrated they could act as recipients for xenogeneic cells. Together, these data hold great promise for the establishment of donor independent liver-humanized rats.

                                                                                                                                                      

        

         

 

Xijun Song ,  Ph.D.         

Beijing Vitalstar Biotechnology Co., Ltd.  

Presentation Title:

Applications of chimeric mouse with human immune system and liver in drug research and development

Abstract:

In the study, we have developed the highest immunodeficiency NPG(NOD‐Prkdcscid Il2rg^null) mouse which accept human tissue and cells very well. Hemopoietic stem cell isolated from human fetal liver and cord blood can reconstitute human immune system in NPG mouse for immune related drug research and development; PDX and CDX model can be generated by transplanted patient derived xenograft or tumor cell line, quickly and efficiently, providing in vivo model for new immune therapy against tumor. We also developed an inducible uPA mouse model on Rag2^nullIl2rg^null background termed URG mice. Transplanted human liver cells reconstitute mouse liver up to 80%, supporting HBV, HCV infection for related drug in vivo test. The humanized URG mouse is also ideal model for drug metabolism and drug toxicity.